Inducible nitric oxide synthase expression and erythropoietin production in human hepatocellular carcinoma cells.

We have previously reported an interaction of nitric oxide (NO) and cyclic 3',5'-guanosine monophosphate (cGMP) in erythropoietin (Epo) production. Further studies have been carried out to clarify the role of NO in the hypoxic regulation of Epo production in Epo producing human hepatocellular carcinoma (Hep3B) cells, which produce Epo in response to physiological stimuli. Our reverse transcriptase-polymerase chain reaction (RT-PCR) technique revealed the expression of iNOS mRNA in Hep3B cells after incubation under hypoxic (1% O2) conditions for 6 hr. Hypoxia also significantly increased medium levels of nitrite in Hep3B cells. In order to investigate the role of NO in Epo production in Hep3B cells under normoxic (20% O2) conditions, we have studied the effects of interferon-gamma (IFN-gamma) on Epo production. IFN-gamma is known to induce iNOS and enhance the production of NO. IFN-gamma produced significant increases in medium levels of Epo and nitrite. IFN-gamma also significantly increased cGMP levels in Hep3B cells. Furthermore, NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, significantly decreased IFN-gamma induced elevations in medium levels of Epo and nitrite as well as cGMP levels in Hep3B cells. These results provide further support for an important role of the NO/cGMP system in hypoxic regulation of Epo production in Hep3B cells.